Speaker: Kacy Richmond Advisor: Dr. Jun Ren (School of Pharmacy) The Positive Effects of Tauroursodeoxycholic Acid on High-Fat Diet-Induced Cardiomyocyte Dysfunction and Endoplasmic Reticulum Stress Speaker: Kacy Richmond Advisor: Dr. Jun Ren (School of Pharmacy) April 19, 2010

Obesity and Endoplasmic Reticulum (ER) Stress Obesity (induced by high-fat diet) is closely associated with the activation of cellular stress signaling and inflammatory pathways. The ER plays a crucial role in the cellular stress response.

Endoplasmic Reticulum (ER) The ER is a membranous network that functions in the synthesis and processing of secretory and membrane proteins.

Obesity and Endoplasmic Reticulum (ER) Stress Numerous conditions, including aging and obesity, can lead to the disruption of ER homeostasis and result in the accumulation of unfolded or misfolded proteins in the ER lumen. This often leads to cell death.

Endoplasmic Reticulum (ER) Stress ER Stress induction Obesity Glucose intolerance Excess lipid accumulation Insulin resistance Mechanical stress Increased mechanical stress Endoplasmic Reticulum (ER) Stress

ER stress signaling (TUDCA) Bip Figure 1. ER stress signaling. The endoplasmic reticulum (ER) is an organelle which synthesizes various secretory and membrane proteins. These proteins are correctly folded and assembled by chaperones in the ER. During stressful conditions such as upon an increase in the misfolded protein level, the chaperones become overloaded and the ER fails to fold and export newly synthesized proteins, leading to ER stress [1–5]. Once ER stress is provoked in the cells, various pathways are activated: phosphorylation of a subunit of translation initiation factor 2 (eIF2a), activation of c-Jun N-terminal kinase (JNK), and splicing of X-box-binding protein-1 (XBP-1). PERK: pancreatic endoplasmic reticulum kinase, IRE1: inositol requiring 1, ATF6: activating transcription factor 6, CHOP: C/EBP homologus protein. (TUDCA)

Tauroursodeoxycholic Acid (TUDCA) Taurine-conjugated derivative of ursodeoxycholic acid (TUDCA) is a potent chemical chaperone that inhibits ER stress. TUDCA administration to obese and diabetic mice normalized hyperglycemia; restored systemic insulin sensitivity; and enhanced insulin action in skeletal muscle. Nevertheless, whether or not TUDCA has an effect on obesity- induced cardiac contractile dysfunction is not clear.

Hypothesis ER stress chaperone — tauroursodeoxycholic acid (TUDCA) – can alleviate cardiac contractile dysfunction induced by high-fat diet (HFD).

Methods High-fat diet induced obesity model: Isolated cardiomyocyte contractile functions; Western blot analysis of ER stress proteins C57 mice LF (10%) Saline HF (45%) TUDCA, ip, 300mg/kg/d 20 weeks Thirty aged-matched C57 male mice will be used in this study. The mice were housed in clear plastic cages in a temperature- and humidity-controlled environment with a 12 hr-light/dark cycle (lights on at 7 AM) and maintained on an ad libitum diet of lab chow and water. Animals were handled in accordance with The Guideline of Animal Care of University of Wyoming. The animals will be fed either with normal laboratory diet (10% of total calorie) or high fat diet (45% of total calorie). Body weights and food intake will be assessed weekly. After 5 months, the animals are further divided into three group randomly based on the changes in body weights. The grouping of the animals is as follows: Group 1 is low fat control group, will receive normal saline intrapetoneally for 15 days; Group 2 is low fat control group with TUDCA treatment (300mg/kg.bw.day-1) for 15 days. Group 3 is high fat diet control group will be given normal saline intraperitoneally for 15 days; Group 4 is high fat diet with TUDCA treatment group (300mg/kg.bw.day-1) for 15 days. 45% HFD mimicks the Western diet 15 days

Isolated cardiomyocyte contractile function Cell length (µm) 100 105 TPS TR90 -dL/dt (maximal velocity of shortening and relengthening) +dL/dt Peak shortening (PS) (Time to peak shortening) (Time to return) This is a typical trace of isolated cardiomyocyte shortening upon electrical stimulus. Let me briefly introduce related parameters. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), the amplitude myocytes shortened upon electrical stimulation, indicative of peak ventricular contractility; time-to-PS (TPS), the duration of myocyte shortening, indicative of systolic duration; time-to-90% relengthening (TR90), the duration to reach 90% relengthening, indicative of diastolic duration (90% rather 100% relengthening was used to avoid noisy signal at baseline level); and maximal velocities of shortening/relengthening, maximal slope (derivative) of shortening and relengthening phases, indicative of maximal velocities of ventricular pressure increase/decrease.

General characteristics of the mice B. C. D. Clue: Obesity>>> ER stress. We are looking at heart function under a prediabetic condition .In our case, it is the HFD induced obesity>> mechanical stress (pressure overload) BMI>30. HW/BW increase >>>cardiac hypertrophy

2. TUDCA partially restores cardiomyocyte function B. C. D. *p<0.05 vs C57-LF; #p<0.05 vs C57-HF; *p<0.05 vs C57-LF; #p<0.05 vs C57-HF;

TUDCA inhibits ER stress

TUDCA inhibits ER stress

TUDCA inhibits ER stress

ER Stress Signaling Pathway Summary HFD Control p-PERK? p-IREα p-cJun CHOP p-JNK TUDCA Treatment Cardiac contractile dysfunction Restored cardiac contractile function p-eIFα C57 HF TUDCA-HF Bip C57 HF TUDCA-HF C57 HF TUDCA-HF p-eIFα

Results, Summary and Conclusion HFD Obesity Glucose intolerance Excess lipid accumulation Insulin resistance Mechanical stress Endoplasmic Reticulum (ER) Stress Cardiomyocyte contractile Dysfunction, Ca 2+ abnormalities TUDCA Cardiomyocyte contractile function

Future directions Study the impact of TUDCA on cardiac insulin signaling Effects of TUDCA on cardiomyocyte function using tunicamycin-induced ER stress in vitro

Acknowledgment Dr. Jun Ren Subat Turdi

Thank you!